The invention generally relates to platinum compounds, which may be used for detectably labeling biological target molecules. More particularly, the invention relates to platinum-based linker compounds, labeling compounds, and detectably labeled molecular probes and methods of using such compounds for the detection and localization of biological substances of interest.
Platinum (coordination) compounds have been considered interesting molecules for a very long time. For a review of some of these compounds and their uses we refer to Reedijk et al., J. Struct. Bonding, 67: 53-72 (1987). This article describes the anti-tumor compound cis-Pt(NH.sub.3).sub.2 Cl.sub.2, which compound has a high affinity for certain biological or bio-organic molecules, including amongst others, proteins and DNA molecules. In particular, it appears that such compounds have a marked affinity for the N.sup.7 -nitrogen atom in the purine bases guanine and adenine, as well as for sulfur groups in macromolecules.
By dissociation of the two chlorine ligands in these compounds, two reactive sites arise, with which such platinum compounds can cross-link within or between molecules. For example, such compounds are known to cross-link between two neighboring guanine and/or adenine bases in the same or opposite DNA strands, thereby inhibiting the replication of the DNA molecules. The application of cis-platinum (cisplatin) as an anti-tumor drug (cytostaticum) is based on this mechanism.
Other applications of the cross-linking capacity of such compounds have been explored. For example, U.S. Pat. No. 4,490,543 discloses platinum-based radiation sensitizers which are said to be derivatives of cisplatin. These sensitizers contain two reactive groups and, accordingly, cross-link biological molecules such as DNA.
In contrast, monochlorinated platinum compounds like Pt(dien)Cl appear to keep their DNA affinity, but they do not form cross-links and interfere only slightly with the base pairing of complementary DNA strands. As such, these compounds have no anti-tumor activity.
According to U.S. Pat. No. 4,711,955, non-radioactive nucleic acid labeling techniques are preferred in present medical-biological practice, especially diagnostic practice. The presently applied known non-radioactive labeling techniques for DNA and RNA are globally to be divided in two categories:
1. Labeling which proceeds via enzymatic or organic synthetic routes; for instance biotin, bromodeoxyuridine (BrdU), digoxigenin, fluorescein and peroxidase; PA1 2. Labeling by direct chemical coupling, like photobiotin, AAF, mercury, and sulfone groups. The '955 document discloses one such labeling method, but does not provide any information concerning use of platinum-based compounds for this purpose. PA1 PROBE is a probe biomolecule for associating to a target biomolecule, PA1 M is a detectable marker moiety, and PA1 X and Y are stabilizing substituents. PA1 M is a detectable marker moiety, PA1 A is a displaceable leaving group, and PA1 X and Y are stabilizing substituents. PA1 PROBE is a biomolecule as defined previously for associating to a target biomolecule, PA1 SUBSTRATE is a substantially solid material, and PA1 X and Y are stabilizing substituents as defined previously. PA1 A and B are the same or different reactive moieties, and PA1 X and Y are stabilizing substituents. PA1 providing to a testable system a platinum-based detectably labeled probe as described above, and PA1 detecting a target biomolecule by determining the extent of association or binding between the detectably labeled probe and the target biomolecule. In this method, the detectably labeled probe binds at least selectively or preferentially to the target biomolecule. In preferred embodiments, however, the detectably labeled probe binds specifically, more preferably uniquely, to the target biomolecule. A highly preferred embodiment involves the induction and detection of in situ hybridization between a detectably labeled nucleic acid of the invention and a target nucleic acid. PA1 contacting a sample, which contains a target biomolecule, with a platinum-based binding substance as described elsewhere herein. In this embodiment, the platinum-based binding substance, which is linked to a substantially solid substrate or surface, possesses a probe molecule associates with or binds to a target biomolecule. Thus, the invention provides, in a preferred situation, a method for specifically binding a target molecule in a quantitative or semi-quantitative assay such as an ELISA. Alternatively, the invention provides a method for separating a target molecule from a sample, such as for purification of the target substance. PA1 PROBE is a probe biomolecule for associating to a target biomolecule, PA1 M is a detectable marker moiety, and PA1 X and Y are stabilizing substituents; and PA1 A and B are the same or different reactive moieties, and PA1 X and Y are stabilizing substituents; and PA1 A is a reactive moiety, PA1 M is a detectable marker moiety, and PA1 X and Y are stabilizing substituents; and PA1 modifying a starting material, which includes a compound of the structure: ##STR11## wherein C represents an electronegative reactive moiety, to produce a platinum-based linker compound of the structure: ##STR12## wherein X represents any stabilizing bridge and wherein A and B represent the same or different reactive moieties. Preferably, X represents a polyamine, more preferably, an aliphatic diamine, still more preferably, an aliphatic diamine having 2-6 carbon atoms, and most preferably, an ethylenediamine group. The reactive moieties A and B are preferably the same. Moreover, A and B are preferably selected from among halogens, NO.sub.3.sup.--, and SO.sub.3.sup.--. Preferably the C moiety is selected from among halogens, NO.sub.3.sup.--, and SO.sub.3.sup.--. PA1 reacting a starting material of the structure: ##STR13## wherein C represents a halogen, with ethylenediamine, and reacting the resulting compound with AgNO.sub.3, to provide a linker comprises a platinum compound of the formula: ##STR14## wherein X represents an ethylenediamine group and wherein A and B represent NO.sub.3.sup.--. PA1 modifying a compound of the structure: ##STR15## wherein X represents any stabilizing bridge and wherein A and B represent the same or different reactive moieties, to produce a platinum-based labeling compound of the structure: ##STR16## wherein M is a detectable marker moiety. PA1 reacting a platinum-based labeling compound of the formula: ##STR17## with a biomolecule, whereby, as a result of said reacting, the A group of the platinum-based labeling compound is replaced by the biomolecule. Preferably, the biomolecule is selected from among proteins, peptides, DNA molecules, and RNA molecules. PA1 a platinum-based linker compound of the formula ##STR18## a detectable marker moiety; and a container. The diagnostic is kit preferably further includes additional detection means such as other marker moieties. PA1 PROBE is a probe biomolecule for associating to a target biomolecule, PA1 M is a detectable marker moiety, and PA1 X and Y are stabilizing substituents. PA1 M is a detectable marker moiety as defined previously, PA1 A is a displaceable leaving group, and PA1 X and Y are stabilizing substituents as defined previously. PA1 PROBE is a biomolecule as defined previously for associating to a target biomolecule, PA1 SUBSTRATE is a substantially solid material, and PA1 X and Y are stabilizing substituents as defined previously. PA1 A and B are the same or different reactive moieties, and PA1 X and Y are stabilizing substituents as defined previously. PA1 1. Direct and almost instantaneous labeling of macromolecules without necessity of enzymatic or organo-synthetic procedures; PA1 2. One-step purification of labeled molecules by means of a simple routine technique; PA1 3. Direct and/or indirect detection of labeled molecules by way of almost all known (microscopic) techniques. PA1 (a) hybridizing a nucleic acid probe labeled with a compound of Formula 1 or 2 with a nucleic acid to be detected; and PA1 (b) detecting the nucleic acid probe. PA1 (a) reacting a compound having the structure: ##STR27## with potassium iodide (KI) in a suitable solvent under suitable conditions so as to form a iodated platinum compound having the structure: ##STR28## (b) reacting the iodated platinum compound obtained in step (a) with ethylenediamine in a suitable solvent so as to form a diethyleneamine iodated platinum compound and represented by the formula Pt(en)I.sub.2 and having the structure: ##STR29## (c) reacting the compound obtained in step (b) with silver nitrate (AgNO.sub.3), the reaction being carried out in a suitable solvent, under suitable conditions so as to form a compound having the structure: ##STR30## (d) reacting the compound obtained in step (c) with potassium chloride (KCl) in a suitable solvent under suitable conditions so as to form a compound having the structure: ##STR31## (e) reacting the compound obtained from step (d) with AgNO.sub.3 in a suitable solvent, under suitable conditions so as to form a compound having the structure: ##STR32## (f) recovering the compound obtained from step (e) as a modified platinum starting compound for the synthesis of marker-bound Pt-containing compounds for use in detectably labeling biological target molecules.
Application of detectable labels to biomolecules has been typically accompanied by a number of problems. Such problems are a result of several factors, including the complexity of previous labeling procedures, limitations on the length of the synthetic oligonucleotides which have been desired to be labeled, the necessity of using health-injuring compounds, and a lack of stability of the label when it has been bound to the nucleic acid.
Given these limitations in the conventional methods of labeling bio-organic target molecules, it is desirable to provide labeling method which overcomes these problems. Probe compounds are desired which can accommodate ready attachment of a large variety of detectable markers. It is also desirable to provide detectably labeled probe compounds which can quickly and easily identify specific bio-organic target molecules such as nucleic acids and proteins. In addition, it would be desirable to provide detectably-labeled probe compounds which would specifically interact with particular bio-organic target molecules, but which do not substantially interfere with their function.
The present invention now provides platinum-containing compounds, in the application of which the above-mentioned disadvantages are effectively removed.